We have cloned five ATP-gated P2X receptor channels (P2XRs) from the pituitary gland: P2X2R, P2X3R, P2X4R, P2X6R, and P2X7R. Our ongoing work is focused on characterization of the roles of these channels in signaling and secretion in secretory pituitary cells and on the structural-functional characterization of recombinant channels. Recently, we showed by quantitative RT-PCR that mRNA transcripts for the P2X4 subunit are the most abundant in rat anterior pituitary tissue and confirm the P2X4R protein expression by Western blot analysis. Single-cell patch-clamp recordings show that extracellular ATP induced an inward depolarizing current in a majority of thyrotropin-releasing hormone-responsive pituitary cells, which resembled the current profile generated by recombinant P2X4R. The channels were activated and desensitized in a dose-dependent manner and deactivated rapidly. Activation of these channels led to stimulation of electrical activity and promotion of voltage-gated and voltage-insensitive calcium influx. In the presence of ivermectin, a specific allosteric modulator of P2X4Rs, there was an approximately fourfold increase in the maximum amplitude of the ATP-induced inward current, accompanied by an increase in the sensitivity of receptors for ATP, slowed deactivation of receptors, and enhanced ATP-induced prolactin release. These results indicate that thyrotropin-releasing hormone-responsive cells, including lactotrophs, express homomeric and/or heteromeric P2X4R, which facilitate calcium influx and hormone secretion. Mammalian P2X receptors contain ten conserved cysteine residues in their ectodomains, which form five disulfide bonds (SS1-5). In collaboration with Dr. Zemkova, we analyzed the relevance of these SS pairs in rat P2X4 receptor function by replacing one or both cysteines with alanine or threonine, expressing receptors in HEK293 cells and studying their responsiveness to ATP in the absence and presence of ivermectin. Response to ATP was not altered when both cysteines forming the SS3 bond (C132-C159) were replaced with threonines. Replacement of SS1 (C116-C165), SS2 (C126-C149) and SS4 (C217-C227), but not SS5 (C261-C270), cysteine pairs with threonines resulted in decreased sensitivity to ATP and faster deactivation times. The maximum current amplitude was reduced in SS2, SS4 and SS5 double mutants and could be partially rescued by ivermectin in SS2 and SS5 double mutants. This response pattern was also observed in numerous single residue mutants, but receptor function was not affected when the 217-cysteine was replaced with threonine or arginine or when the 261-cysteine was replaced with alanine. These results suggest that the SS1, SS2 and SS4 bonds contribute substantially to the structure of the ligand binding pocket, while the SS5 bond located towards the transmembrane domain contributes to receptor gating. Our collaborative work with P. Catholic University of Chile, was focused on allosteric modulation of P2X2R by several compounds, mainly acting at receptors ectodomain. Like copper, mercury, a metal that induces oxidative stress in cells, also stimulates the activity of P2X2R and inhibits the activity of P2X4R. However, the mercury modulation is not related to the extracellular residues critical for copper modulation. To identify the site(s) for mercury action, we generated two chimeras using the full size P2X2 subunit, termed P2X2a, and a splice variant lacking a 69-residue segment in the C-terminal, termed P2X2b, as donors for intracellular and transmembrane segments and the P2X4 subunit as the donor for ectodomain segment of chimeras. The potentiating effect of mercury on ATP-induced current was preserved in Xenopus oocytes expressing P2X4/2a chimera, but was absent in oocytes expressing P2X4/2b chimera. Site directed mutagenesis experiments revealed that the Cys-430 residue mediates effects of mercury on the P2X2aR activity. Because mercury could act as an oxidative stress inducer, we also tested whether hydrogen peroxide and mitochondrial stress inducers myxothiazol, and rotenone mimicked mercury effects. These experiments revealed that these compounds potentiated the ATP-evoked P2X2aR and P2X4/2aR currents, but not P2X2bR and P2X2a-C430A and P2X2a-C430S mutant currents, whereas antioxidants dithiothreitrol and N-acetylcysteine prevented the hydrogen peroxide-potentiation. Alkylation of Cys-430 residue with methylmethane-thiosulfonate also abolished the mercury and hydrogen peroxide potentiation. Altogether, these results are consistent with the hypothesis that the Cys-430 residue is an intracellular P2X2aR redox sensor. In collaboration with Dr. Ambudkars group, we also studied dependence of multidrug resistance protein-mediated cyclic nucleotide efflux on the background sodium conductance in pituitary cells. These cells fire action potentials and release cyclic nucleotides both spontaneously and in response to agonist stimulation, but the relationship between electrical activity and cyclic nucleotide efflux has not been studied previously. We showed that a tetrodotoxin-resistant background sodium conductance was critical for firing of action potentials and that multidrug resistance proteins (MRPs) MRP4 and MRP5 contribute to cyclic nucleotide efflux. We also showed that abolition of the background sodium conductance in rat pituitary cells by complete or partial replacement of extracellular sodium with organic cations or sucrose induced a rapid and reversible hyperpolarization of cell membranes and inhibition of action potential firing, accompanied by a rapid inhibition of cyclic nucleotide efflux. Valinomycin-induced hyperpolarization of plasma membranes also inhibited cyclic nucleotide efflux, whereas depolarization of cell membranes induced by the inhibition of calcium influx or stimulation of sodium influx by gramicidin was accompanied by a facilitation of cyclic nucleotide efflux. In contrast, inhibition of cyclic nucleotide efflux by probenecid did not affect the background sodium conductance. In human embryonic kidney 293 cells stably transfected with human MRP4 or MRP5, replacement of bath sodium with organic cations also hyperpolarized the cell membranes and inhibited cyclic nucleotide efflux. In these cells, the sodium/hydrogen antiporter monensin did not affect the membrane potential and was practically ineffective in altering cyclic nucleotide efflux. In both pituitary and MRP4- and MRP5-expressing cells, 3-3-2-(7-chloroquinolin-2-yl)vinylphenyl-(2-dimethylcarbamoylethylsulfanyl)methylsulfanyl propionic acid (MK571) inhibited cyclic nucleotide efflux. These results indicate that the MRP4/5-mediated cyclic nucleotide efflux can be rapidly modulated by membrane potential determined by the background sodium conductance. Finally, in collaboration with Dr. Naors group we studied mechanisms of the reciprocal cross talk between gonadotropin-releasing hormone (GnRH) and prostaglandin receptors. Results of these investigations indicate that GnRH stimulates arachidonic acid release via the calcium-independent phospholipase A2 and not via the more common calcium-dependent cytosolic phospholipase A2. GnRH stimulates COX-1 and COX-2 expression via the protein kinase C-Src/phosphatidylinositol 3-kinase/MAPK pathway. COX-2 transcription is mediated apparently by the nuclear factor-B and the CCAAT/enhancer-binding protein sites. The documented inhibition by PGF2 and PGI2 of the autoregulation of GnRH receptor expression is calcium elevation, cAMP formation, and MAPK activation.